Protein Mass-Modulated Effects in Alkaline Phosphatase

Area of Science:

• Biochemistry
• Enzymology
• Protein Dynamics

Background:

• Isotopically substituted proteins (heavy proteins) reveal mass-dependent effects on enzyme catalysis.
• Femtosecond protein dynamics are hypothesized to link to transition-state barrier crossing.
• Previous studies on deuterium-labeled alkaline phosphatase (AP) suggested a significant kinetic isotope effect.

Purpose of the Study:

• To investigate the role of heavy enzyme protein dynamics in AP-catalyzed reactions.
• To characterize the steady-state and chemical step properties of native, deuterated, and fully labeled AP.
• To determine if AP's catalytic mechanism is influenced by altered protein mass.

Main Methods:

• Preparation of native AP, [2H]AP, and [2H,13C,15N]AP.
• Measurement of steady-state kinetic parameters (kcat, Km, kchem).
• Analysis of single-turnover rate constants.

Main Results:

• Deuterated and fully labeled APs exhibited unaltered steady-state and single-turnover rate constants compared to native AP.
• Alkaline phosphatase demonstrates minimal mass-dependent effects on its catalytic efficiency.
• The catalytic mechanism of AP appears independent of protein isotopic composition.

Conclusions:

• Alkaline phosphatase (AP) is an enzyme where catalysis is not significantly linked to protein mass.
• Mass-independent catalysis in AP is likely due to reactant-linked atomic motions and the conserved mass of catalytic zinc ions.
• The findings contrast with other enzymes showing mass-dependent kinetic isotope effects.